Extending the Lifetime of Native GTP-Bound Ras for Site-Resolved NMR Measurements: Quantifying the Allosteric Dynamics

摘要

Characterization of native GTP-bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras.GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras.GDP signals, is applied to the site-resolved NMR measurement of the allosteric dynamics of Ras.GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor-binding pockets, but the motions are more confined than those of Ras.GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos-based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.